MAP kinases in the mammalian circadian system--key regulators of clock function

Over the past 7 years, there has been spectacular progress in our understanding of the molecular basis of the circadian pacemaker in many species, from yeast to mammals. However, the biochemical signalling mechanisms that underpin synchronization of the clock to environmental cues are still poorly understood. Recently, attention has been focused on the role of mitogen-activated protein (MAP) kinase in biological timekeeping. It has been proposed that signal transduction via the MAP kinase cascades allows environmental information to be assimilated intracellularly within the circadian clock to produce changes in the phasing of clock gene expression, which, in turn, underlies clock-controlled phase-resetting of biological rhythms. This review examines the evidence for MAP kinase, particularly extracellular regulated kinases 1/2, involvement in the circadian clock and looks at the putative upstream regulators and downstream substrates of this signalling system.     

Mapping Full-Length Porcine Endogenous Retroviruses in a Large White Pig

Xenotransplantation may bridge the widening gap between the shortage of donor organs and the increasing number of patients waiting for transplantation. However, a major safety issue is the potential cross-species transmission of porcine endogenous retroviruses (PERV). This problem could be resolved if it is possible to produce pigs that do not contain replication-competent copies of this virus. In order to determine the feasibility of this, we have determined the number of potentially replication-competent full-length PERV proviruses and obtained data on their integration sites within the porcine genome. We have screened genomic DNA libraries from a Large White pig for potentially intact proviruses. We identified six unique PERV B proviruses that were apparently intact in all three genes, while the majority of isolated proviruses were defective in one or more genes. No intact PERV A proviruses were found in this pig, despite the identification of multiple defective A proviruses. Genotyping of 30 unrelated pigs for these unique proviruses showed a heterogeneous distribution. Two proviruses were uncommon, present in 7 of 30 and 3 of 30 pigs, while three were each present in 24 of 30 pigs, and one was present in 30 of 30 animals examined. Our data indicate that few PERV proviruses in Large White pigs are capable of productive infection and suggest that many could be removed by selective breeding. Further studies are required to determine if all potentially functional proviruses could be removed by breeding or whether gene knockout techniques will be required to remove the residuum.     

Cross-talk between pRb/E2F and Wnt/beta-catenin pathways: E2F1 induces axin2 leading to repression of Wnt signalling and to increased cell death

The pRb/E2F and Wnt/beta-catenin pathways are two of the most frequently deregulated pathways in human cancers. In this study, we show that E2F1 up-regulates the expression of axin2. Further, we show that axin2 can repress Wnt signalling leading to reduced cell growth and increased cell death. This represents cross-talk between major pathways involved in the formation of tumours. We use our data to suggest a novel mechanism for tumour suppression.     

African Glucose-6-Phosphate Dehydrogenase Alleles Associated with Protection from Severe Malaria in Heterozygous Females in Tanzania

X-linked Glucose-6-phosphate dehydrogenase (G6PD) A- deficiency is prevalent in sub-Saharan Africa populations, and has been associated with protection from severe malaria. Whether females and/or males are protected by G6PD deficiency is uncertain, due in part to G6PD and malaria phenotypic complexity and misclassification. Almost all large association studies have genotyped a limited number of G6PD SNPs (e.g. G6PD202 / G6PD376), and this approach has been too blunt to capture the complete epidemiological picture. Here we have identified 68 G6PD polymorphisms and analysed 29 of these (i.e. those with a minor allele frequency greater than 1%) in 983 severe malaria cases and controls in Tanzania. We establish, across a number of SNPs including G6PD376, that only female heterozygotes are protected from severe malaria. Haplotype analysis reveals the G6PD locus to be under balancing selection, suggesting a mechanism of protection relying on alleles at modest frequency and avoiding fixation, where protection provided by G6PD deficiency against severe malaria is offset by increased risk of life-threatening complications. Our study also demonstrates that the much-needed large-scale studies of severe malaria and G6PD enzymatic function across African populations require the identification and analysis of the full repertoire of G6PD genetic markers.     

Polyploid speciation inHedera (Araliaceae): Phylogenetic and biogeographic insights based on chromosome counts and ITS sequences

Variation in chromosome number and internal transcribed sequences (ITS) of nrDNA is used to infer phylogenetic relationships of a wide range ofHedera species. Polyploidy was found to be frequent inHedera, with diploid, tetraploid, hexaploid and octoploid populations being detected. Nucleotide additivity occurs in the ITS sequences of one tetraploid (H. hibernica) and two hexaploid species (H. maderensis, H. pastuchovii), suggesting that all three species originated by allopolyploidisation. ITS sequence polymorphism and nucleotide characters may indicate the presence of an ancient genome persistent only in some allopolyploid species. Phylogenetic analyses of ITS sequence data reveal two lineages ofHedera: one containing all sequences belonging to extant diploids plus the tetraploidH. algeriensis, and a second that includes this ancient ITS type and others exclusive to several polyploid species. The origin of the polyploids is evaluated on the basis of morphology, chromosome counts, ITS sequence polymorphism, and phylogenetic analyses. Reconstruction of reticulate evolution inHedera agrees with two allopolyploid areas on both sides of the Mediterranean basin. Morphological, molecular and cytological evidence also suggests an active dispersal ofHedera populations that may account for three independent introductions in Macaronesia.     

The biochemical properties and phylogenies of phosphofructokinases from extremophiles

The enzyme phosphofructokinase (PFK) is a defining activity of the highly conserved glycolytic pathway, and is present in the domains Bacteria, Eukarya, and Archaea. PFK subtypes are now known that utilize either ATP, ADP, or pyrophosphate as the primary phosphoryl donor and share the ability to catalyze the transfer of phosphate to the 1-position of fructose-6-phosphate. Because of the crucial position in the glycolytic pathway of PFKs, their biochemical characteristics and phylogenies may play a significant role in elucidating the origins of glycolysis and, indeed, of metabolism itself. Despite the shared ability to phosphorylate fructose-6-phosphate, PFKs that have been characterized to date now fall into three sequence families: the PFKA family, consisting of the well-known higher eukaryotic ATP-dependent PFKs together with their ATP- and pyrophosphate-dependent bacterial cousins (including the crenarchaeal pyrophosphate-dependent PFK of Thermoprotetus tenax) and plant pyrophosphate-dependent phosphofructokinases; the PFKB family, exemplified by the minor ATP-dependent PFK activity of Escherichia coli (PFK 2), but which also includes at least one crenarchaeal enzyme in Aeropyrum pernix; and the tentatively named PFKC family, which contains the unique ADP-dependent PFKs from the euryarchaeal genera of Pyrococcus and Thermococcus, which are indicated by sequence analysis to be present also in the methanogenic species Methanococcus jannaschii and Methanosarcina mazei.     

Interactive effects of elevated CO2, N deposition and climate change on extracellular enzyme activity and soil density fractionation in a California annual grassland

Elevated CO₂, N deposition and climate change can alter ecosystem‐level nutrient cycling both directly and indirectly. We explored the interactive effects of these environmental changes on extracellular enzyme activity and organic matter fractionation in soils of a California annual grassland. The activities of hydrolases (polysaccharide‐degrading enzymes and phosphatase) increased significantly in response to nitrate addition, which coincided with an increase in soluble C concentrations under ambient CO₂. Water addition and elevated CO₂ had negative but nonadditive effects on the activities of these enzymes. In contrast, water addition resulted in an increase in the activities of lignin‐degrading enzymes (phenol oxidase and peroxidase), and a decrease in the free light fraction (FLF) of soil organic matter. Independent of treatment effects, lignin content in the FLF was negatively correlated with the quantity of FLF across all samples. Lignin concentrations were lower in the aggregate‐occluded light fraction (OLF) than the FLF, and there was no correlation between percent lignin and OLF quantity, which was consistent with the protection of soil organic matter in aggregates. Elevated CO₂ decreased the quantity of OLF and increased the OLF lignin concentration, however, which is consistent with increased degradation resulting from increased turnover of soil aggregates. Overall, these results suggest that the effects of N addition on hydrolase activity are offset by the interactive effects of water addition and elevated CO₂, whereas water and elevated CO₂ may cause an increase in the breakdown of soil organic matter as a result of their effects on lignin‐degrading enzymes and soil aggregation, respectively.     

RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

Here, we show that Escherichia coli Ribonuclease III cleaves specifically the RNA genome of hepatitis C virus (HCV) within the first 570 nt with similar efficiency within two sequences which are ~400 bases apart in the linear HCV map. Demonstrations include determination of the specificity of the cleavage sites at positions C²⁷ and U³³ in the first (5′) motif and G⁴³⁹ in the second (3′) motif, complete competition inhibition of 5′ and 3′ HCV RNA cleavages by added double-stranded RNA in a 1:6 to 1:8 weight ratio, respectively, 50% reverse competition inhibition of the RNase III T7 R1.1 mRNA substrate cleavage by HCV RNA at 1:1 molar ratio, and determination of the 5′ phosphate and 3′ hydroxyl end groups of the newly generated termini after cleavage. By comparing the activity and specificity of the commercial RNase III enzyme, used in this study, with the natural E.coli RNase III enzyme, on the natural bacteriophage T7 R1.1 mRNA substrate, we demonstrated that the HCV cuts fall into the category of specific, secondary RNase III cleavages. This reaction identifies regions of unusual RNA structure, and we further showed that blocking or deletion of one of the two RNase III-sensitive sequence motifs impeded cleavage at the other, providing direct evidence that both sequence motifs, besides being far apart in the linear RNA sequence, occur in a single RNA structural motif, which encloses the HCV internal ribosome entry site in a large RNA loop.     

Synchronization of Firing in Cortical Fast-Spiking Interneurons at Gamma Frequencies: A Phase-Resetting Analysis

Fast-spiking (FS) cells in the neocortex are interconnected both by inhibitory chemical synapses and by electrical synapses, or gap-junctions. Synchronized firing of FS neurons is important in the generation of gamma oscillations, at frequencies between 30 and 80 Hz. To understand how these synaptic interactions control synchronization, artificial synaptic conductances were injected in FS cells, and the synaptic phase-resetting function (SPRF), describing how the compound synaptic input perturbs the phase of gamma-frequency spiking as a function of the phase at which it is applied, was measured. GABAergic and gap junctional conductances made distinct contributions to the SPRF, which had a surprisingly simple piecewise linear form, with a sharp midcycle break between phase delay and advance. Analysis of the SPRF showed how the intrinsic biophysical properties of FS neurons and their interconnections allow entrainment of firing over a wide gamma frequency band, whose upper and lower frequency limits are controlled by electrical synapses and GABAergic inhibition respectively.